ABSTRACT Besides eliciting adaptive immune responses, there are many anti-viral factors expressed in host cells to combat viral infection. The recent discovery of numerous such factors indicates that most viral replication steps are kept in check by host defense mechanisms. Of course, viruses evolve to escape from these mechanisms. Therefore, the history of viral evolution is a constant cat-and-mouse game between human anti-viral defense mechanisms and viral escape mutants. Nevertheless, studying these cellular anti-viral mechanisms is extremely important for developing potent and specific anti-HIV therapies. We recently observed that the human piwi-like RNA-mediated gene silencing 2 (Hili) protein strongly inhibits HIV replication. Hili's anti-HIV effect occurs at the step of viral protein synthesis (translation) by binding and depleting several tRNAs corresponding to amino acid codons rarely used in the human genome but frequently found in HIV. In addition, levels of Hili mRNA expression are increased upon stimulation via the T-cell receptor, indicating that Hili is a cellular anti-viral factor responding to T-cell activation. Based on these results, we hypothesize that human Piwi-family proteins are new potent anti-HIV factors expressed in CD4+ T cells. The goal of the proposed study is to understand the anti-HIV mechanism of Piwi-family proteins and evaluate their roles in the maintenance of HIV cellular resistance to new HIV infection and replication. Also, the mechanism whereby Piwi expression is regulated in CD4+ T cells will be further explored. Finally, several new approaches for effectively inhibiting HIV replication by depleting these tRNAs will be developed and validated. The proposed study will lead to a development of new anti-HIV strategies by manipulating viral protein expression.